Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) which is designated PrPSc. A chromosomal gene encodes both the cellular prion protein (PrPC) as well as PrPSc. Pulse-chase experiments with scrapie-infected cultured cells indicate that PrPSc is formed by a post-translational process. PrP is translated in the endoplasmic reticulum, modified as it passes through the Golgi, and is transported to the cell surface. Release of nascent PrP from the cell surface by phosphatidylinositol-specific phospholipase C or hydrolysis with dispase prevented PrPSc synthesis. At 18 degrees C, the synthesis of PrPSc was inhibited under conditions that other investigators report a blockage of endosomal fusion with lysosomes. Our results suggest that PrPSc synthesis occurs after PrP transits from the cell surface. Whether all of the PrP molecules have an equal likelihood to be converted into PrPSc or only a distinct subset is eligible for conversion remains to be established. Identifying the subcellular compartment(s) of PrPSc synthesis should be of considerable importance in defining the molecular changes that distinguish PrPSc from PrPC.
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PMID:Evidence for synthesis of scrapie prion proteins in the endocytic pathway. 135 61

A common feature of scrapie and related transmissible spongiform encephalopathies is the accumulation of an abnormal protease-resistant form of PrP which may be the major component of the infectious agent. While it is known that both the normal (protease-sensitive) PrP and protease-resistant PrP are encoded by the same endogenous gene, the nature of the disease-associated modification of PrP is not understood. To study the cellular events leading to the formation of protease-resistant PrP, we have compared its biosynthesis to that of its normal isoform in scrapie-infected mouse neuroblastoma cells. In pulse-chase labeling experiments, the protease-resistant PrP was synthesized and degraded much more slowly than the normal PrP, suggesting that protease-resistant PrP is made from a protease-sensitive precursor. More significantly, we found that the precursor of protease-resistant PrP was eliminated from intact cells by treatments with phosphatidylinositol-specific phospholipase C and trypsin. This demonstrated that, unlike the protease-resistant PrP itself, the precursor is phospholipase- and protease-sensitive and at least transiently found on the cell surface. By these criteria, the precursor of protease-resistant PrP is indistinguishable from the normal PrP isoform. These results indicate that the conversion of PrP to the protease- and phospholipase-resistant state is a post-translational event that occurs after the precursor reaches the cell surface.
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PMID:The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. 168 Aug 59

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.
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PMID:Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. 169 23

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.
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PMID:Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. 171 82

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.
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PMID:Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells. 196 4

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.
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PMID:Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. 196 66

The scrapie agent protein (Sp33-37 or PrPSc) is the disease-associated isoform of a normal cellular membrane protein (Cp33-37 or PrPC) of unknown function. We report that normal human lymphocytes and lymphoid cell lines, but not erythrocytes or granulocytes, express PrPC mRNA and protein. PrPC is detectable on the surface of lymphocytes; the surface immunoreactivity is sensitive to phosphatidylinositol-specific phospholipase C, indicating glycosyl-phosphatidylinositol membrane anchorage. Lymphocyte PrPC surface abundance is increased by cell activation, and polyclonal antibodies to PrPC suppress mitogen-induced activation. We conclude that PrPC is a lymphocyte surface molecule that may participate in cell activation.
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PMID:Cellular isoform of the scrapie agent protein participates in lymphocyte activation. 196 32

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.
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PMID:Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. 197 60

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.
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PMID:Scrapie prion protein contains a phosphatidylinositol glycolipid. 244 40

Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.
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PMID:Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells. 256 14


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